Chiral resolution of profoxydim: Implications for herbicidal efficacy and ecotoxicological assessment

Chiral pesticides may exhibit stereoselective differences in biological activity, toxicity, and environmental behaviour, making the assessment of individual stereoisomers increasingly important for improving pesticide efficacy and reducing environmental risks. Profoxydim, a widely used cyclohexanedione herbicide for rice cultivation, contains two chiral centres and is commercialized as a mixture of four stereoisomers, although the biological and ecotoxicological properties of each stereoisomer remain largely unknown. A robust analytical method was developed for the quantification and isolation of the four profoxydim stereoisomers. Complete stereoisomeric separation was achieved under normal-phase high performance liquid chromatography (HPLC) conditions using a Chiralcel OJ-H column with a heptane/ethanol (20:80) mobile phase containing 0.05% formic acid at 0.70 mL/min and 25 °C. This approach enabled, for the first time, the isolation of all four stereoisomers for subsequent biological and ecotoxicological assessment. The herbicidal activity of the racemic mixture and each isolated stereoisomer was evaluated through seed germination assays. Root growth was substantially more sensitive to profoxydim than coleoptile growth, with a 1 mg/L dose markedly inhibiting root elongation. Dose‒response analyses revealed clear stereoselectivity, with isomers 1 and 3 exhibiting significantly higher herbicidal activity (IC₅₀ values of 0.02 and 0.01 mg/L, respectively). To assess the potential effects on nontarget aquatic organisms, in vitro cytotoxicity assays using RTG-2 rainbow trout cells and Microtox tests with Vibrio fischeri were conducted. Small stereoselective differences were observed in fish cell cytotoxicity, with all the stereoisomers showing lower toxicity than racemic profoxydim (IC₅₀ = 342 mg/L). However, the Microtox assay revealed lower toxicity for the racemate (EC₅₀ = 48 mg/L) than for the individual stereoisomers, indicating that the stereoisomeric composition may influence microbial toxicity. Overall, these results demonstrate distinct stereoselective patterns in the herbicidal activity and ecotoxicity of profoxydim. Identifying the most active and least toxic stereoisomers may contribute to reducing application rates and environmental risks while maintaining effective weed control.